Non-canonical heterogeneous cellular distribution and co-localization of CaMKII\u03b1 and CaMKII\u03b2 in the spinal superficial dorsal horn

Max Larsson

doi:10.1007/s00429-017-1566-0

Abstract

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key enzyme in long-term plasticity in many neurons, including in the nociceptive circuitry of the spinal dorsal horn. However, although the role of CaMKII heterooligomers in neuronal plasticity is isoform-dependent, the distribution and co-localization of CaMKII isoforms in the dorsal horn have not been comprehensively investigated. Here, quantitative immunofluorescence analysis was used to examine the distribution of the two major neuronal CaMKII isoforms, α and β, in laminae I–III of the rat dorsal horn, with reference to inhibitory interneurons and neuronal populations defined by expression of parvalbumin, calretinin, and calbindin D28k. Unexpectedly, all or nearly all inhibitory and excitatory neurons showed both CaMKIIα and CaMKIIβ immunoreactivity, although at highly variable levels. Lamina III neurons showed less CaMKIIα immunoreactivity than laminae I–II neurons. Whereas CaMKIIα immunoreactivity was found at nearly similar levels in inhibitory and excitatory neurons, CaMKIIβ generally showed considerably lower immunoreactivity in inhibitory neurons. Distinct populations of inhibitory calretinin neurons and excitatory parvalbumin neurons exhibited high CaMKIIα-to-CaMKIIβ immunoreactivity ratios. CaMKIIα and CaMKIIβ immunoreactivity showed positive correlation at GluA2+ puncta in pepsin-treated tissue. These results suggest that, unlike the forebrain, the dorsal horn is characterized by similar expression of CaMKIIα in excitatory and inhibitory neurons, whereas CaMKIIβ is less expressed in inhibitory neurons. Moreover, CaMKII isoform expression varies considerably within and between neuronal populations defined by laminar location, calcium-binding protein expression, and transmitter phenotype, suggesting differences in CaMKII function both between and within neuronal populations in the superficial dorsal horn.

Highlights

Ca2?/calmodulin-dependent protein kinase II (CaMKII) is well established as a pivotal enzyme in neuronal plasticity (Hell 2014; Lisman et al 2012; Coultrap and Bayer 2012)
The CaMKII antibodies used have been well characterized and shown to be specific for the respective isoform in the brain (e.g., Bachstetter et al 2014; Ding et al 2013; van Woerden et al 2009), antibody specificity tests should preferentially be performed on the tissue of interest, as antigen cross-reactivity may be observed in only certain types or regions of tissue (Larsson et al 2011)
CaMKIIblike immunoreactivity (CaMKIIb-LI) was weak in wildtype mouse spinal cord sections, possibly because the tissue was too strongly fixed, and heat-mediated antigen retrieval was, used. This yielded a pattern of CaMKIIb-LI in wild-type mouse spinal cord similar to that in rat spinal cord not subjected to antigen retrieval (Fig. 1b; see below); an additional weak immunolabeling over myelin was observed

Summary

Introduction

Ca2?/calmodulin-dependent protein kinase II (CaMKII) is well established as a pivotal enzyme in neuronal plasticity (Hell 2014; Lisman et al 2012; Coultrap and Bayer 2012). Most studies on the role of CaMKII in neuronal plasticity have focused on the a subunit or used tools that do not differentiate between different isoforms. Major functional differences between CaMKIIa and CaMKIIb have been identified (Liu and Murray 2012; Hell 2014). The b isoform binds F-actin and targets a/b heteromers to dendritic spines (e.g., Borgesius et al 2011; Shen et al 1998); this binding is abolished by Ca2?/calmodulin or autophosphorylation, allowing the holoenzyme to translocate to the postsynaptic density in an activity-dependent manner (Shen and Meyer 1999).

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